The present invention relates to methods for identifying and characterizing compounds that modulate the activity of a member of the EDG family of receptors, including EDG-1, EDG-2, EDG-3, EDG-4, and EDG-5 (gene sequences and encoded amino acid sequences shown in SEQ ID NOS. 4, 1, 5, 6, and 7, respectively), and a similar receptor named PSP-24 (gene sequence and encoded amino acid sequence shown in SEQ ID NOS. 8 and 9).
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. Frequently, binding of a ligand to a cell-surface receptor represents the first step in a cascade of events that results in a cellular response. The ligands recognized by specific receptors include a diverse array of molecules such as peptides, deoxyribonucleotide triphosphates and phospholipids.
Research into phospholipid signaling is an area of intense scientific investigation, as more and more bioactive lipids are being identified and their actions characterized. One important addition to the growing list of lipid messengers is lysophosphatidic acid (1-acyl-2-hydroxy-sn-glycero-3-phosphate, LPA), the simplest of all glycerophospholipids. While LPA has long been known as a precursor of phospholipid biosynthesis in both eukaryotic and prokaryotic cells, only recently has LPA emerged as an intercellular signaling molecule that is rapidly produced and released by activated cells, notably platelets, to influence target cells by acting on a specific cell-surface receptor. Moolenaar (1994) Trends Cell Biol. 4:213-219. Besides being synthesized and processed to more complex phospholipids in the endoplasmic reticulum, LPA can be generated through the hydrolysis of pre-existing phospholipids following cell activation. The best documented example concerns thrombin-activated platelets, where LPA production is followed by its extracellular release. Eichholtz et al. (1993) Biochem. J. 291:677-680. Platelet LPA is formed, at least in large part, through phospholipase A2 (PLA2)-mediated deacylation of newly generated phosphatidic acid (PA). Gerrard and Robinson (1989) Biochim. Biophys. Acta 1001:282-285. Distinct PA-specific PLA2 activity has been identified in platelets (Ca2+-dependent) and in rat brain (Ca2+-independent), but little is known about its mode of regulation. Billah et al. (1981) J. Biol. Chem. 256:5399-5403; and Thompson and Clark (1995) Biochem. J. 306:305-309.
It remains to be examined at what stage of the platelet activation response LPA is produced and how it is released into the extracellular environment. Given the wide variety of LPA responsive cell types, LPA production and release are unlikely to be restricted to platelets. Indeed, there is preliminary evidence that growth factor-stimulated fibroblasts can also produce LPA. Fukami and Takenawa (1992) J. Biol. Chem. 267:10988-10993. Furthermore, LPA may be formed and released by injured cells, probably due to nonspecific activation of phospholipases. Many other cell systems remain to be examined for LPA production.
In freshly prepared mammalian serum, LPA concentrations are estimated to be in the range of approximately 2-20 xcexcM, with oleoyl- and palmitoyl-LPA being the predominant species. Tokumura et al. (1994) Am. J. Physiol. 267:C204-C210; and Eichholtz et al. (1993) Biochem. J. 291:677-680. LPA is not detectable in platelet-poor plasma, whole blood, or cerebrospinal fluid. Tigyi and Miledi (1992) J. Biol. Chem. 267:21360-21367. In common with long chain fatty acids, LPA binds with high affinity to serum albumin at a molar ratio of about 3:1. Tigyi et al. (1991) J. Biol. Chem. 266:20602-20609; Thumser et al. (1994) Biochem. J. 301:801-806. It is notable that serum albumin contains several other, as yet unidentified lipids (methanol-extractable) with LPA-like biological activity. Tigyi and Miledi (1992) J. Biol. Chem. 267:21360-21367. This raises the interesting possibility that LPA may belong to a new family of phospholipid mediators showing overlapping biological activities and acting on distinct receptors; conceivably, the ether-linked phospholipid platelet-activating factor (PAF) and the mitogenic lipid sphingosine 1-phosphate may also belong to this putative family. Zhang et al. (1991) J. Cell Biol. 114:155-167.
The range of biological responses to LPA is quite diverse, ranging from induction of cell proliferation to stimulation of neurite retraction and even slimemold chemotaxis, and the body of knowledge continues to grow as more and more cellular systems are tested for LPA responsiveness. Jalink et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:1857-1861; Jalink et al. (1993) Cell Growth and Differ. 4:247-255; and Moolenaar (1995) Curr. Opin. Cell Biol. 7:203-210; Dyer et al. (1992) Molec. Brain Res. 14:293-301; Dyer et al. (1992) Molec. Brain Res. 14:302-309; Tigyi and Miledi (1992) J. Biol. Chem. 267:21360-21367.
Although its precise physiological and pathological functions in vivo remain to be explored, LPA derived from platelets has all the hallmarks of an important mediator of wound healing and tissue regeneration. Thus, in addition to acting as an autocrine stimulator of platelet aggregation, LPA stimulates the growth of fibroblasts, vascular smooth muscle cells, endothelial cells, and keratinocytes. Moolenaar (1994) Trends Cell Biol. 4:213-219; Jalink et al. (1994) Biochim. Biophys. Acta 1198:185-196; Van Corven et al. (1989) Cell 59:45-54; Tigyi et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:1908-1912; Tokumura et al. (1994) Am. J. Physiol. 267:C204-C210; and Piazza et al. (1995) Exp. Cell Res. 216:51-64. Intriguingly, it has been observed that LPA acts as an inhibitor of eukaryotic DNA polymerase xcex1. Murakami-Murofushi et al. (1992) J. Biol. Chem. 267:21512-21517. LPA also exhibits anti-mitogenic activity toward myeloma cells, presumably through a distinct receptor subtype. Tigyi et al. (1994) Proc. Natl. Acad. Sci. 91:1908-1912; Murakami-Murofushi et al. (1993) Cell Structure and Function 18:363-370.
In addition to stimulating cell growth and proliferation, LPA promotes cellular tension and cell-surface fibronectin binding, which are important events in wound repair and regeneration. Zhang et al. (1994) J. Cell Biol. 127:1447-1459; Kolodney et al. (1993) J. Biol. Chem. 268:23850-23855; and Lapetina et al. (1981) J. Biochem. 256:5037-5040. As a product of the blood-clotting process, LPA is a normal constituent of serum (but not platelet-poor plasma), where it is present in an albumin-bound form at physiologically relevant concentrations. Tigyi and Miledi (1992) J. Biol. Chem. 267:21360-21367; and Eichholtz et al. (1993) Biochem. J. 291:677-680.
Recently, anti-apoptotic activity has also been ascribed to LPA. PCT Application No. PCT/US94/13649. In this study, an actively proliferating cell line was rescued from serum withdrawal-induced apoptosis by LPA. In another study, evidence has been presented suggesting that LPA can suppress apoptosis in vitro as well as in ischemic organs such as heart and liver. Wu et al. (1996) Transplantation (in press).
Apoptosis is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death may be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation, and infection by human immunodeficiency virus (HIV). Wyllie (1980) Nature 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun. 118:392-399; Duke and Cohen (1986) Lymphokine Res. 5:289-299; Tomei et al. (1988) Biochem. Biophys. Res. Commun. 155:324-331; Kruman et al. (1991) J. Cell. Physiol. 148:267-273; Ameisen and Capron (1991) Immunol. Today 12:102-105; and Sheppard and Ascher (1992) J. AIDS 5:143-147. Agents that affect the biological control of apoptosis thus have therapeutic utility in numerous clinical indications.
Cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage characterize Apoptotic cell death. Gerschenson et al. (1992) FASEB J. 6:2450-2455; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267-293. The blebs, small, membrane-encapsulated spheres that pinch off of the surface of apoptotic cells, may continue to produce superoxide radicals which damage surrounding cell tissue and may be involved in inflammatory processes.
While apoptosis is a normal cellular event, pathological conditions and a variety of injuries can also induce it. Apoptosis is involved in a wide variety of conditions, including, but not limited to, cardiovascular disease; cancer regression; immune disorders, including, but not limited to, systemic lupus erythematosus; viral diseases; anemia; neurological disorders; diabetes; hair loss; rejection of organ transplants; prostate hypertrophy; obesity; ocular disorders; stress; aging; and gastrointestinal disorders, including, but not limited to, diarrhea and dysentery. In the myocardium, apoptotic cell death follows ischemia and reperfusion.
In Alzheimer""s disease, Parkinson""s disease, Huntington""s chorea, epilepsy, amyotrophic lateral sclerosis, stroke, ischemic heart disease, spinal cord injury and many viral infections, for example, abnormally high levels of cell death occur. In at least some of these diseases, there is evidence that the excessive cell death occurs through mechanisms consistent with apoptosis. Among these are 1) spinal cord injury, where the severing of axons deprives neurons of neurotrophic factors necessary to sustain cellular viability; 2) stroke, where after an initial phase of necrotic cell death due to ischemia, the rupture of dead cells releases excitatory neurotransmitters such as glutamate and oxygen free radicals that stimulate apoptosis in neighboring healthy neurons; and 3) HIV infection, which induces apoptosis of T-lymphocytes.
In contrast, the level of apoptosis is decreased in cancer cells, which allows the cancer cells to survive longer than their normal cell counterparts. As a result of the increased number of surviving cancer cells, the mass of a tumor can increase even if the doubling time of the cancer cells does not increase. Furthermore, the high level of expression in a cancer cell of the bcl-2 gene, which is involved in regulating apoptosis and, in some cases, necrotic cell death, renders the cancer cell relatively resistant to chemotherapeutic agents and to radiation therapy.
There is considerable evidence of plasma membrane receptors for LPA. LPA-binding proteins have been reported in mammalian tissues and labeled using a photoaffinity crosslinker derivative. Liliom et al. (1996) Am. J. Physiol. 270:C772-C778; Thomson et al. (1994) Mol. Pharmacol. 45:718-723; and van der Bend et al. (1992) EMBO J. 11:2495-2501. In X. laevis oocytes, LPA elicits oscillatory Clxe2x88x92 currents. Tigyi and Miledi (1992) J. Biol. Chem. 267:21360-21367. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways.
G proteins are comprised of three subunits: a guanyl-nucleotide binding xcex1 subunit; a xcex2 subunit; and a xcex3 subunit. G proteins cycle between two forms, depending on whether GDP or GTP is bound thereto. When GDP is bound the Gxcex1xcex2xcex3 protein exists as an inactive heterotrimer, the Gxcex1xcex2xcex3 complex. When GTP is bound the xcex1 subunit dissociates, leaving a Gxcex2xcex3 complex. Importantly, when a Gxcex1xcex2xcex3 complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and, hence, the rate of dissociation of the bound the xcex1 subunit from the Gxcex2xcex3 complex increases. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena.
At least four G protein-mediated signaling pathways have been identified in the action of LPA. These are: 1) stimulation of phospholipase C and phospholipase D; 2) inhibition of adenylyl cyclase; 3) activation of Ras and the downstream Raf/MAP kinase pathway; and 4) tyrosine phosphorylation of focal adhesion proteins in concert with remodeling of the actin cytoskeleton in a Rho-dependent manner.
GTP-binding proteins fall into two broad classes of regulatory proteins; the heterotrimeric G-proteins, and small GTPases. The alpha subunit of heterotrimeric G-proteins (Gxcex1) and the small GTPases, as typified by the proto-oncogene Ras, share certain structural homology, and cycle between an active GTP-bound state and an inactive GDP-bound state. When stimulated by an appropriate signal, G-proteins and small GTPases become activated by the binding of GTP and physically interact with effector molecules to transduce the signal to the cell. In the case of G-proteins, binding of GTP to the xcex1 subunit causes the low molecular weight Gxcex1 to dissociate from the Gxcex2xcex3 dimer where either Gxcex1 or Gxcex2xcex3 can act as the signal transducer. An intrinsic GTPase activity hydrolyses GTP to GDP and thus attenuates the signal. Ancillary proteins collectively known as exchange factors are responsible for replacing GDP for GTP and reactivating the GTP-binding protein. Heterotrimeric G-protein coupled receptors are a special class of receptors. It is estimated that G-protein coupled receptors comprise 0.1% of the human genome (including olfactory and visual receptors) which could place the number of different receptors in the thousands. The common structural feature of these receptors are seven hydrophilic membrane spanning domains. Based on the three dimensional model of bacterial rhodopsin, it is predicted that the receptors would form a barrel shaped structure with the ligand binding domains being the extracellular loops and/or the transmembrane domains.
Recently, three putative receptors for LPA have been identified suggesting that functionally different LPA receptors may exist that dictate the particular cellular response of LPA. Hecht, J. H., et al. (1996) J. Cell. Biol. 135(4), 1071-1083; Macrae, A. D., et al. (1996) Mol. Brain. Res. 42, 245-254; An, S., et al. (1997) Biochm. Biophys. Res. Com. 231, 619-622; Guo, Z., et al. (1996) Proc. Natl. Acad. Sci. USA 93, 14367-14372; An, et al., J. Biol. Chem. (1998). Most cell types respond to LPA making it difficult to characterize the receptor dependency of a particular response to LPA since the response cannot be solely attributed to a single LPA receptor. In particular, it is difficult to assess ligand binding specificity of an LPA receptor without a naive cell line because other lipid receptors may exist with overlapping ligand specificity. Therefor, the yeast Saccharomyces cerevisiae was used to study the human LPA receptor EDG-2 (or Vzg-1). S. cerevisiae contain no endogenous LPA receptors and is therefore a potentially useful organism in which to functionally express LPA receptors and analyze their ligand specificity. Other mammalian receptors have been functionally expressed in Saccharomyces including the sommatostatin receptor. (Price, L. A., et al. (1995) Mol. Cell. Biol. 15(11), 6188-6195), the A2a adenosine receptor (Price, L. A., et al. (1996) Mol. Pharmacol. 50(4), 829-837) and the xcex22-adrenergic receptor (King, K., et al., (1990) Science 250, 121-123).
FIG. 1 shows a detailed schematic of the yeast pheromone-inducible MAP Kinase cascade. Saccharomyces contains a single heterotrimeric G-protein that is activated by mating factor binding to a specific receptor. Blumer, K. J., and Thorner, J. (1990) Proc. Natl. Acad. Sci. USA 87, 4363-4367. Upon stimulation by an occupied receptor, the a subunit of the heterotrimeric G protein (Gxcex1, the GPA1 gene product (Dietzel, C., and Kurjan, J. (1987) Cell 50, 1001-1010; Miyajima, I., et al. (1987) Cell 50, 1011-1019) becomes bound to GTP and dissociates from the xcex2xcex3 dimer. In yeast, it is the xcex2xcex3 dimer that transduces the signal to Ste 11 (the MEKK equivalent (Lange-Carter, C. A., et al. (1993) Science 260, 315-319)) and Ste7 (the MEK equivalent (Neiman, A. M., and Herskowitz, I. (1994) Proc. Natl. Acad. Sci. USA 91, 3398-3402)). The active GTP-bound version of Gxcex1 is inactivated by hydrolysis of GTP to GDP at which time, Gxcex1 can re-associate with Gxcex2xcex3 and attenuates the signal (Blinder, D., and Jenness, D. D. (1989) Mol. Cell. Biol. 9, 3720-3726; Cole, G., (1990) Mol. Cell. Biol. 10(510-517); Dietzel, C., and Kurjan, J. (1987) Cell 50, 1001-1010; Miyajima, I., et al. (1987) Cell 50, 1011-1019). Like the mammalian MAP kinase, the yeast MAP kinases Fus1 and Kss1 activate a transcriptional activator, the STE12 gene product (Elion, E. A., et al. (1994) Mol. Biol. Cell 4, 495-510). Activated Ste12 in turn activates the transcription of several mating-inducible genes such as FUS1 (Elion, E. A., et al. (1991) Cold Spring Harbor Symp. Quant. Biol. 56, 41-49; Peter, M., et al. (1993) Cell 73, 747-760). To study the EDG-2 receptor using the yeast pheromone response pathway system, a strain carrying a mutation in the FAR1 gene was used. This mutation has the effect of uncoupling the MAP kinase cascade from cell cycle arrest allowing the yeast to continue growing during MAP kinase activation (Peter, M., et al. (1993) Cell 73, 747-760; Peter, M., and Herskowitz, I. (1994) Science 265, 1228-1231). Secondly, a mutationally inactivated SST2 gene was created to increase the sensitivity of the strain to G-protein activation. The SST2 gene encodes a GTPase activating protein (GAP) for the Gxcex1 subunit (the GPA1 gene product) (Dohlman, H. G., et al. (1996) Mol. Cell. Biol. 16(9), 5194-5209). By inactivating the SST2 gene product, Gxcex1 remains in the GTP-bound state longer and thus increases the steady-state concentration of the signal transducing xcex2xcex3 dimer. Finally, to quantify the response, the bacterial lacZ gene was fused to the mating inducible FUS1 promoter to create a reporter gene.
The ubiquitous presence of the response elicited by LPA in almost every cell line tested, combined with the amphiphilic character of LPA that makes radioligand binding assays extremely difficult, has presented considerable difficulties in the molecular cloning of its receptors. In view of the potential physiological significance of LPA receptors in terms of wound healing, cell regeneration and cell proliferation and apoptosis, there is a need for drug screening assays exhibiting increased specificity that facilitate the search for agonists, inverse agonists, or antagonists of LPA, as well as methods for screening analogues of LPA to determine their ability to activate EDG-2, for elucidating the pharmacological properties of these proteins.
The present invention addresses this need. Herein are described methods of screening for agonists or antagonists of EDG-1, EDG-2, EDG-3, EDG-4, and EDG-5, as well as methods of counter screening for agonists or antagonists that are specific for only one of these EDG receptors.
All references cited herein are incorporated by reference in their entirety.
Methods of screening for pharmaceutical agents that stimulate, as well as pharmaceutical agents that inhibit, EDG-1, EDG-2, EDG-3, EDG-4, and EDG-5 activity are provided.
The present invention encompasses a method for identifying compounds which modulate the activity of any of the EDG receptors, comprising the steps of: a) contacting recombinant host cells, modified to contain the DNA of SEQ. ID. NO. 1, 4, 5, 6, 7 or 8, which is operably linked to control sequences for expression, with at least one compound or signal whose ability to modulate the activity of the EDG receptor is sought to be determined, and b) analyzing the cells for a difference in functional response mediated by said receptor. More specifically, the present invention encompasses contacting said cells with at least one composition whose ability to modulate the activity of said receptor is sought to be determined, and monitoring said cells for a change in the level of a particular signal associated with activation of the EDG receptor. EDG receptors encompassed by the present invention include EDG-1, EDG-2, EDG-3, EDG-4, and EDG-5. An additional receptor encompassed by the present invention is PSP-24, a receptor of LPA discovered in mice, which can be used as a screen to evaluate the specificity of a particular ligand for any of the EDG family of receptors. For purposes of the present discussion, PSP-24 shall be encompassed by the expressions xe2x80x9cEDG family of receptorsxe2x80x9d and xe2x80x9cEDG related receptors, because it has similarities, including being an LPA receptor.
Additionally, the present invention contemplates a method for modulating the signal transduction activity of the EDG receptor, comprising contacting said receptor with an effective amount of at least one compound identified by the method described above.
The present invention also encompasses an agonist, antagonist, inverse agonist, or allosteric modulator identified by the above methods.
In an alternative embodiment, the present invention encompasses a method for detecting an agonist, antagonist, inverse agonist, or allosteric modulator of an EDG receptor having activity comprising the steps of: a) exposing a compound to an EDG receptor coupled to a response pathway, under conditions and for a time sufficient to allow interaction of the compound with the EDG receptor and an associated response through the pathway, and b) detecting an increase or a decrease in the stimulation of the response pathway resulting from the interaction of the compound with the EDG receptor, relative to the absence of the tested compound and therefrom determining the presence of an agonist, antagonist, inverse agonist, or allosteric modulator.
In yet another embodiment, the present invention encompasses a method for detecting an LPA agonist, antagonist, inverse agonist, or allosteric modulator of LPA receptor comprising the steps of a) exposing a compound to the EDG-2 receptor coupled to a response pathway, under conditions and for a time sufficient to allow interaction of the compound with the EDG-2 receptor and an associated response through the pathway, and b) detecting an increase or a decrease in the stimulation of the response pathway resulting from the interaction of the compound with the EDG-2 receptor, relative to the absence of the tested compound and therefrom determining the presence of an agonist, antagonist, inverse agonist, or allosteric modulator.
In yet another embodiment, the invention encompasses a method for detecting inverse agonists of LPA, comprising the steps of a) exposing a compound and LPA to the EDG-2 receptor coupled to a response pathway, under conditions and for a time sufficient to allow interaction of LPA with the EDG-2 receptor and an associated response through the pathway, and b) detecting an increase or a decrease in the stimulation of the response pathway, relative to the absence of the tested compound and therefrom determining the presence of an inverse agonist of LPA. In yet another embodiment of the present invention, a method of detecting compounds that modulate the interaction between a ligand of an EDG related receptor and the EDG related receptor is encompassed, comprising: exposing a labeled ligand of an EDG related receptor to a cell expressing said EDG related receptor; exposing a labeled compound that is believed to interact with an EDG related receptor to said cell, and detecting a change in the amount of labeled ligand bound to said cell.